Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

Background

Power Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.

Methodology/Principal Findings

PBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.

Conclusions/Significance

Our data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.     

A regulatory effect of IL-21 on T follicular helper-like cell and B cell in rheumatoid arthritis

Introduction

Interleukin (IL)-21 is a member of type I cytokine family. Recent studies indicate that IL-21 can promote T follicular helper (Tfh) cell differentiation and survival, a specialized T cell subset which provides help for B cell. It can also regulate the activation, proliferation and differentiation of human B cell and immunoglobulin (Ig) production as well as isotype switching of plasma cell. Rheumatoid arthritis (RA) is characterized by auto-antibodies overproduction such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody, suggesting a pivotal role of Tfh cell and B cell in the pathogenesis of RA. This study aimed to investigate whether IL-21 had a regulatory effect on Tfh cell and B cell in RA.

Methods

Serum IL-21 concentrations were measured by ELISA. The correlations between serum IL-21 levels and clinical features of RA patients were analyzed by Spearman''s rank test. The percentages of Tfh-like cells, IL-21 receptor (R) expression on Tfh-like cells and B cells in peripheral blood (PB) were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated by rIL-21 (100 ng/ml) in the presence or absence of anti-CD40 and/or anti-IgM, and changes of IL-21R, activation-associated surface markers (CD25, CD69 and CD40), the proliferation, apoptosis and differentiation of B cells were analyzed by flow cytometry. Production of IgG and IgM in the culture supernatants was determined by ELISA.

Results

The results showed that the serum IL-21 levels in RA patients were significantly higher than that of healthy controls (HC). IL-21 concentrations were positively correlated with 28-joint count disease activity score (DAS28) and anti-CCP antibody in RA patients with high IL-21 levels. Furthermore, the frequencies of peripheral CXCR5⁺PD-1⁺CD4⁺ Tfh-like cells markedly increased in RA patients and the percentages of Tfh-like cells were positively correlated with DAS28 and anti-CCP antibody levels. Moreover, elevated IL-21 levels were also correlated with the frequencies of Tfh-like cells. IL-21R expression on both Tfh-like cells and B cells were significantly enhanced in RA patients. In cultures vitro, exogenous IL-21 upregulated IL-21R expression and activation-associated surface markers on B cells and promoted more B cell proliferation in RA than in HC. This IL-21-mediated effect could be reversed by IL-21R-specific neutralizing antibody. Importantly, IL-21 promoted more differentiation of B cell into plasmablast and higher levels of IgG and IgM production in RA than in HC.

Conclusions

Increased serum IL-21 levels in RA patients correlate with DAS28, anti-CCP antibody and frequencies of Tfh-like cells. IL-21 supports B cell activation, proliferation and antibody secretion via IL-21R pathway. Thus, IL-21 may be involved in the pathogenesis of RA and antagonizing IL-21 could be a novel strategy for the therapy of RA.     

Kinetics of T helper subsets and associated cytokines correlate well with the clinical activity of graft-versus-host disease

Background

CD4⁺interferon (IFN)-γ⁺ T cell (Th1) and CD4⁺interleukin (IL)-4⁺ T cell (Th2) polarizations are crucial in the pathogenesis of graft-versus-host disease (GVHD). However, this hypothesis is largely based on animal experiments of Parent-into-F1 GVHD model. The causal relationship between kinetics of Th1, Th2 and associated cytokines and the clinical activity of GVHD in a real world situation remains unknown.

Methodology

Peripheral blood was collected every week prospectively from Day 0 to Day 210 (patients without GVHD) or Day 300 (patients with chronic GVHD) after allogeneic peripheral blood stem cell transplantation in consecutive 27 patients. The frequencies of Th1 and Th2 within CD4⁺ T cells were determined by flow cytometry and pplasma IFN-γ, IL-12, IL-4, and IL-10 were determined by ELISA.

Principal Findings

Kinetics of Th1, Th2 frequency, and the plasma IL-10 and IFN-γ more commonly coincided with, rather than predicted, the activity of GVHD. These markers are significantly higher when acute or chronic GVHD developed. The kinetics of IL-10 is especially correlated well with the activity of GVHD during clinical course of immunosuppressive treatment. For patients with hepatic GVHD, there is a positive correlation between plasma IL-10 levels and the severity of hepatic injury. The frequency of Th2 is also significant higher in acute GVHD and tends to be higher in chronic GVHD. Interestingly, there is a very good positive correlation between the frequency of Th1 and Th2 (r = 0.951, p<0.001). The plasma level of IL-4 and IL-12 are not associated with the activity of GVHD.

Conclusions

The frequency of Th1, Th2 within CD4⁺ T cells and plasma IL-10 and IFN-γ are good biomarkers of GVHD. Plasma IL-10 can also be used to monitor the therapeutic responsiveness. Furthermore, both Th1 and Th2 likely contribute to the pathogenesis of GVHD.     

Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity,immune regulation and therapy

Introduction

In autoimmune diseases, IL-17 producing T-cells (Th17), a pro-inflammatory subset of T-cells, are pathophysiologically involved. There is little knowledge on the role of Th17 cells in granulomatosis with polyangiitis (GPA). In the present study, we investigated Th17 cells, Tregs and subsets of circulating Th17 cells in GPA and related results to disease activity.

Methods

42 GPA patients in remission, 18 with active disease and 14 healthy controls (HC) were enrolled. Th17 cells, their subsets and regulatory T-cells were determined by intracellular fluorescence activated cell sorter (FACS). Data are given as mean percentage ±SD of total T-helper-cells.

Results

Th17 cells are expanded in active and quiescent GPA as compared to HC (1.7±1.4% vs. 0.7 ±0.3%, P = 0.006 and 1.9 ±1.5% vs. 0.7 ±0.3%, P<0.0001). Th17 expansion is stable over time and does not decline when remission is achieved. However, a negative association of Th17 cells and steroid dosage is observed (r=-0.46, P = 0.002). The Th17 expansion was not balanced by Tregs as indicated by skewed Th17/Treg ratios in active and quiescent GPA. Th17 subsets co-producing IFNγ or IL-10 are significantly increased in GPA. GPA patients in remission not receiving maintenance therapy have significantly more IL-10/IL-17A double positive T-cells than HC (0.0501 ±0.031% vs. 0.0282 ±0.016%, P = 0.007).

Conclusions

We provide evidence for a persistent, unbalanced expansion of Th17 cells and Th17 subsets which seems to be independent of disease activity. Maintenance therapy reduces -but does not normalize- Th17 expansion.     

The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

Background

Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc.

Methodology and Principal Findings

Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFβ and IFNγ using flow cytometry. Levels of IL-17, IL-6, IL-1α and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNγ and TGFβ were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1α were increased in all or subsets of SSc patients.

Conclusion and Significance

The combination of IL-17, IFNγ and TGFβ levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.     

Elevated Frequencies of Circulating Th22 Cell in Addition to Th17 Cell and Th17/Th1 Cell in Patients with Acute Coronary Syndrome

Background

Atherosclerosis is a chronic inflammatory disease mediated by immune cells. Th22 cells are CD4⁺ T cells that secret IL-22 but not IL-17 or IFN-γ and are implicated in the pathogenesis of inflammatory disease. The roles of Th22 cells in the pathophysiologic procedures of acute coronary syndrome (ACS) remain unclear. The purpose of this study is to investigate the profile of Th22, Th17 and Th17/Th1 cells in ACS patients, including unstable angina (UA) and acute myocardial infarction (AMI) patients.

Design and Methods

In this study, 26 AMI patients, 16 UA patients, 16 stable angina (SA) patients and 16 healthy controls were included. The frequencies of Th22, Th17 and Th17/Th1 cells in AMI, UA, SA patients and healthy controls were examined by flow cytometry. Plasma levels of IL-22, IL-17 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA).

Results

Th22, Th17 and Th17/Th1 cells were significantly increased in AMI and UA patients compared with SA patients and healthy controls. Moreover, plasma IL-22 level was significantly elevated in AMI and UA patients. In addition, Th22 cells correlated positively with IL-22 as well as Th17 cells in AMI and UA patients.

Conclusion

Our findings showed increased frequencies of both Th22 and Th17 cells in ACS patients, which suggest that Th22 and Th17 cells may play a potential role in plaque destabilization and the development of ACS.     

Increased frequencies of Th22 cells as well as Th17 cells in the peripheral blood of patients with ankylosing spondylitis and rheumatoid arthritis

Background

T-helper (Th) 22 is involved in the pathogenesis of inflammatory diseases. The roles of Th22 cells in the pathophysiological of ankylosing spondylitis (AS) and rheumatoid arthritis (RA) remain unsettled. So we examined the frequencies of Th22 cells, Th17 cells and Th1 cells in peripheral blood (PB) from patients with AS and patients with RA compared with both healthy controls as well as patients with osteoarthritis.

Design and Methods

We studied 32 AS patients, 20 RA patients, 10 OA patients and 20 healthy controls. The expression of IL-22, IL-17 and IFN-γ were examined in AS, RA, OA patients and healthy controls by flow cytometry. Plasma IL-22 and IL-17 levels were examined by enzyme-linked immunosorbent assay.

Results

Th22 cells, Th17 cells and interleukin-22 were significantly elevated in AS and RA patients compared with OA patients and healthy controls. Moreover, Th22 cells showed positive correlation with Th17 cells as well as interleukin-22 in AS and RA patients. However, positive correlation between IL-22 and Th17 cells was only found in AS patients not in RA patients. In addition, the percentages of both Th22 cells and Th17 cells correlated positively with disease activity only in RA patients not in AS patients.

Conclusions

The frequencies of both Th22 cells and Th17 cells were elevated in PB from patients with AS and patients with RA. These findings suggest that Th22 cells and Th17 cells may be implicated in the pathogenesis of AS and RA, and Th22 cells and Th17 cells may be reasonable cellular targets for therapeutic intervention.     

A Higher Frequency of Circulating IL-22+CD4+ T Cells in Chinese Patients with Newly Diagnosed Hashimoto’s Thyroiditis

Background

IL-22 and IL-17A are implicated in the pathogenesis of autoimmune diseases. However, the role of IL-22⁺ and IL-17A⁺ CD4⁺ T cells in the pathogenesis of Hashimoto’s thyroiditis (HT) is not fully understood. This study investigates serum IL-22 and IL-17A levels and determines the frequency of circulating IL-22⁺ CD4⁺ T cells in HT patients to understand their roles in the pathogenesis of HT.

Methods

The levels of serum IL-22, IL-17A and IFN-γ and the frequency of circulating IL-22⁺CD4⁺ and IL-17A⁺CD4⁺ T cells in 17 HT patients and 17 healthy controls (HC) were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The levels of serum free triiodothyronine (FT4), free thyroxine (FT3), thyroid stimulating hormone (TSH), anti-thyroid peroxidase (TPO) and anti-thyroglobulin antibodies (TgAb) by chemiluminescent enzyme immunoassay and radioimmunoassay.

Results

The percentages of circulating IL-22⁺CD4⁺ and IL-17⁺CD4⁺ T cells (p<0.0001, p<0.0001) and the levels of serum IL-22, IL-17A and IFN-γ (p<0.0001, p<0.0001, p = 0.0210) in the HT patients were significantly higher than that in the HC. The percentages of IL-22⁺CD4⁺ T cells were positively correlated with Th17 cells (r = 0.8815, p<0.0001) and IL-17A⁺IL-22⁺CD4⁺ T cells (r = 0.8914, p<0.0001), but were negatively correlated with Th1 cells (r = −0.6110, p<0.0092) in the HT patients. The percentages of Th22 cells, Th17 cells and IL-17A⁺IL-22⁺CD4⁺ T cells were negatively correlated with the levels of serum TSH in the HT patients (r = −0.8402, p<0.0001; r = −0.8589, p<0.0001; r = −0.8289 p<0.0001, respectively).

Conclusions

A higher frequency of circulating IL-22⁺CD4⁺ and IL-17A⁺CD4⁺ T cells may be associated with the development of HT in Chinese patients.     

Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3''-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.     

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice

Background

H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking.

Methodology/Principal Findings

Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8⁺ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8⁺ T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8⁺ T cells.

Conclusions/Significance

Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.     

Constitutively altered frequencies of circulating follicullar helper T cell counterparts and their subsets in rheumatoid arthritis

Introduction

Circulating CD4 T cells expressing CXCR5, ICOS and/or PD-1 are counterparts of follicular helper T cells (Tfh). There are three subpopulations of circulating Tfh (cTfh): CXCR5 + CXCR3 + CCR6- (Tfh-Th1), CXCR5 + CXCR3-CCR6- (Tfh-Th2) and CXCR5 + CXCR3-CCR6+ (Tfh-Th17). Our objective was to study the B cell helping capacity of cTfh subsets, and examine their frequency in Rheumatoid Arthritis (RA) patients, together with the frequency of circulating plasmablasts (CD19 + CD20-CD38^high).

Methods

Peripheral blood was drawn from RA patients with active disease (RA-a, DAS28 >2.6) (n = 17), RA in remission (RA-r, DAS28 <2.6) (n = 17) and healthy controls (HC) (n = 34). cTfh and plasmablast frequencies were determined by flow cytometry. Cocultures of sorted CD4 + CXCR5+ T cell subpopulations were established with autologous CD19 + CD27- naïve B cells of HC, and concentrations of IgG, A and M were measured in supernatants.

Results

Isolated Tfh-Th2 and Tfh-Th17 but not Tfh-Th1 cells, induced naïve B cells to secrete IgG and IgA. The frequency of CXCR5+ cells gated for CD4+ T cells was not different among HC, RA-a and RA-r. In contrast, both RA-a and RA-r patients demonstrated an increased frequency of CD4 + CXCR5 + ICOS+ T cells and augmented (%Tfh-Th2 + %Tfh-Th17)/%Tfh-Th1 ratio as compared with HC. In addition, RA-a but not RA-r patients, showed an increased frequency of circulating plasmablasts.

Conclusion

Both RA-a and RA-r patients demonstrate an increased frequency of cTfh and overrepresentation of cTfh subsets bearing a B cell helper phenotype, suggesting that altered germinal center dynamics play a role in RA pathogenesis. In contrast, only RA-a patients show an increased proportion of circulating plasmablasts.     

Decreased interleukin 27 expression is associated with active uveitis in Beh?et’s disease

Instruction

Interleukin 27 (IL-27) is an important regulator of the proinflammatory T-cell response. In this study, we investigated its role in the pathogenesis of Behçet’s disease (BD).

Methods

IL-27 mRNA in peripheral blood mononuclear cells (PBMCs) was examined by performing RT-PCRs. Cytokine levels in sera or supernatants of PBMCs, naïve CD4⁺ T cells, dendritic cells (DCs) and DC/T cells were determined by enzyme-linked immunosorbent assay. We used RNA interference in naïve CD4⁺ T cells to study the role of interferon regulatory factor 8 (IRF8) in the inhibitory effect of IL-27 on Th17 cell differentiation. Flow cytometry was used to evaluate the frequency of IL-17- and interferon γ–producing T cells.

Results

The expression of IL-27p28 mRNA by PBMCs and IL-27 in the sera and supernatants of cultured PBMCs were markedly decreased in patients with active BD. A higher frequency of IL-17-producing CD4⁺ T (Th17) cells and increased IL-17 production under Th17 polarizing conditions were observed in patients with active BD. IL-27 significantly inhibited Th17 cell differentiation. Downregulation of IRF8 by RNA interference abrogated the suppressive effect of IL-27 on Th17 differentiation. IL-27 inhibited the production of IL-1β, IL-6 and IL-23, but promoted IL-10 production, by DCs. IL-27-treated DCs inhibited both the Th1 and Th17 cell responses.

Conclusions

The results of the present study suggest that a decreased IL-27 expression is associated with disease activity in BD patients. Low IL-27 expression may result in a higher Th1 and Th17 cell response and thereby promote the autoinflammatory reaction observed in BD. Manipulation of IL-27 may offer a new treatment modality for this disease.     

Disruption of rhythms of molecular clocks in primary synovial fibroblasts of patients with osteoarthritis and rheumatoid arthritis, role of IL-1��/TNF

Introduction

Circadian rhythms play an important role in the body and in single cells. Rhythms of molecular clocks have not been investigated in synovial fibroblasts (SF) of patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The study was initiated to fill this gap and to study effects of interleukin (IL)-1β/tumor necrosis factor (TNF) on rhythmicity in synovial fibroblasts of RA and OA patients.

Methods

The presence of BMAL-1, CLOCK, Period 1 and Period 2 proteins in synovial tissue was investigated by immunofluorescence. The presence of mRNA of molecular clocks was studied during 72 h by qPCR. Characteristics of rhythms were studied with time series analysis.

Results

BMAL-1, CLOCK, Period 1 and Period 2 proteins were abundantly present in synovial tissue of OA, RA and controls. Receiving synovial tissue at different operation time points during the day (8:00 am to 4:00 pm) did not reveal a rhythm of BMAL-1 or Period 1 protein. In OASF and RASF, no typical rhythm curve of molecular clock mRNA was observed. Time series analysis identified a first peak between 2 and 18 hours after synchronization but a period was not detectable due to loss of rhythm. TNF inhibited mRNA of CLOCK, Period 1 and Period 2 in OASF, while IL-1β and TNF increased these factors in RASF. This was supported by dose-dependently increased levels in MH7A RA fibroblasts. In RASF, IL-1β and TNF shifted the first peak of BMAL-1 mRNA to later time points (8 h to 14 h).

Conclusion

Rhythmicity is not present in primary OASF and RASF, which is unexpected because fibroblasts usually demonstrate perfect rhythms during several days. This might lead to uncoupling of important cellular pathways.     

CD39+ Regulatory T cells suppress generation and differentiation of Th17 cells in human malignant pleural effusion via a LAP-dependent mechanism

Background

Both regulatory T cells (Tregs) and T helper IL-17-producing cells (Th17 cells) have been found to be involved in human malignancies, however, the possible implication of Tregs in regulating generation and differentiation of Th17 cells in malignant pleural effusion remains to be elucidated.

Methods

The numbers of both CD39⁺Tregs and Th17 cells in malignant pleural effusion and peripheral blood from patients with lung cancer were determined by flow cytometry. The regulation and mechanism of Tregs on generation and differentiation of Th17 cells were explored.

Results

Both CD39⁺Tregs and Th17 cells were increased in malignant pleural effusion when compared with blood, and the numbers of CD39⁺Tregs were correlated negatively with those of Th17 cells. It was also noted that high levels of IL-1β, IL-6, and TGF-β1 could be observed in malignant pleural effusion when compared the corresponding serum, and that pleural CD39⁺Tregs could express latency-associated peptide on their surface. When naïve CD4⁺ T cells were cocultured with CD39⁺Tregs, Th17 cell numbers decreased as CD39⁺Treg numbers increased, addition of the anti-latency-associated peptide mAb to the coculture reverted the inhibitory effect exerted by CD39⁺Tregs.

Conclusions

Therefore, the above results indicate that CD39⁺Tregs inhibit generation and differentiation of Th17 cells via a latency-associated peptide-dependent mechanism.     

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Th17 and Th22 cells in psoriatic arthritis and psoriasis

Introduction

The aim of this study was to characterize interleukin 17 (IL-17) and interleukin 22 (IL-22) producing cells in peripheral blood (PB), skin, synovial fluid (SF) and synovial tissue (ST) in patients with psoriasis (Ps) and psoriatic arthritis (PsA).

Methods

Flow cytometry was used to enumerate cells making IL-22 and IL-17, in skin and/or SF and PB from 11 patients with Ps and 12 patients with PsA; skin and PB of 15 healthy controls and SF from rheumatoid arthritis (RA) patients were used as controls. Expression of the interleukin 23 receptor (IL-23R) and chemokine receptors CCR4 and CCR6 was examined. Secretion of IL-17 and IL-22 was measured by ELISA. ST was analysed by immunohistochemical staining of IL-17 and IL-22.

Results

Increased frequencies of IL-17+ and IL-22+ CD4+ T cells were seen in PB of patients with PsA and Ps. IL-17 secretion was significantly elevated in both PsA and Ps, whilst IL-22 secretion was higher in PsA compared to Ps and healthy controls. A higher proportion of the CD4+ cells making IL-17 or IL-22 expressed IL-23R and frequencies of IL-17+, CCR6+ and CCR4+ T cells were elevated in patients with Ps and those with PsA. In patients with PsA, CCR6+ and IL-23R + T cells numbers were elevated in SF compared to PB. Increased frequencies of IL-17+ and IL-22+ CD4+ T cells were demonstrated in Ps skin lesions. In contrast, whilst elevated frequencies of CD4+ IL-17+ cells were seen in PsA SF compared to PB, frequencies of CD4+ IL-22+ T cells were lower. Whereas IL-17 expression was equivalent in PsA, osteoarthritis (OA) and RA ST, IL-22 expression was higher in RA than either OA or PsA ST, in which IL-22 was strikingly absent.

Conclusions

Elevated frequencies of IL-17 and IL-22 producing CD4+ T cells were a feature of both Ps and PsA. However their differing distribution at disease sites, including lower frequencies of IL-22+ CD4+ T cells in SF compared to skin and PB, and lack of IL-22 expression in ST suggests that Th17 and Th22 cells have common, as well as divergent roles in the pathogenesis of Ps and PsA.     

Imbalanced Frequencies of Th17 and Treg Cells in Acute Coronary Syndromes Are Mediated by IL-6-STAT3 Signaling

Aims

Extensive evidence suggests inflammatory components participate in the pathogenic processes of acute coronary syndromes (ACS). In this study, we aimed to elucidate the role and mechanism underlying the imbalance of Th17 and Treg cell peripheral populations in the pathogenesis of ACS.

Methods and Results

Using a flow cytometric analysis, we observed a significantly increased frequency of Th17 cells and a concurrently decreased CD4⁺CD25⁺Foxp3⁺ Treg cells in patients with ACS. To elucidate the mechanism of Th17/Treg imbalance in ACS, 22 inflammatory cytokines were measured using multiplexed immunobead-based assays. Of six elevated cytokines in ACS patients, only IL-6 was positively correlated with a higher Th17 cell level (r = 0.39, P<0.01). Relying on IL-6 stimulating and neutralizing studies, we demonstrated a direct role for IL-6 in sera from ACS patients with an increased frequency of Th17 cells. IL-6 induces the differentiation of Th17 cells from naïve CD4⁺ T cells through STAT3 activation and RORγt induction. However, we observed that high levels of TGF-β1 inhibited IL-6-dependent Th17 cell differentiation, indicating a complex interplay between the two cytokines in the control of Th17 and Treg cell populations.

Conclusions

Our results demonstrate the role of IL-6-STAT3 signaling in ACS through increased Th17 cell differentiation. These findings indicate that IL-6 neutralizing strategies could present novel therapeutic avenues in the treatment of ACS.